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Abstract

Background

Cytokines such as interleukin 6 (IL-6) have been implicated in dual functions in neuropsychiatric
disorders. Little is known about the genetic predisposition to neurodegenerative and
neuroproliferative properties of cytokine genes. In this study the potential dual
role of several IL-6 polymorphisms in brain morphology is investigated.

Methodology

In a large sample of healthy individuals (N = 303), associations between genetic variants of IL-6 (rs1800795; rs1800796, rs2069833, rs2069840) and brain volume (gray matter volume)
were analyzed using voxel-based morphometry (VBM). Selection of single nucleotide
polymorphisms (SNPs) followed a tagging SNP approach (e.g., Stampa algorigthm), yielding
a capture 97.08% of the variation in the IL-6 gene using four tagging SNPs.

Conclusions/significance

These findings suggest a possible neuroprotective role of the G-allele of the SNP
rs1800795 on hippocampal volumes. Studies on the role of this SNP in psychiatric populations
and especially in those with an affected hippocampus (e.g., by maltreatment, stress)
are warranted.

Keywords:

Introduction

Inflammation is implicated in the etiology and pathophysiology of several brain pathologies
(e.g., major depression [1-3], Alzheimer’s disease [4], and post-stroke depression [5]), as well as in cognitive aging [6] and mortality [7]. Specific markers of systemic inflammation such as cytokines have been identified
as important mediators of neurodegenerative [8] and neuroplastic [9,10] processes relevant to neuropsychiatric disorders. Some of these proteins (e.g., interleukin
1 beta, interleukin 6, tumor necrosis factor) play a critical role in physiological
CNS processes, such as cognitive function under immunologically unchallenged conditions
[2,6].

Interleukin 6 (IL-6) is a cytokine that has demonstrated both neurodegenerative [4] and neuroprotective [11,12] properties. For example, increased levels of IL-6 are associated with neuropsychiatric
conditions, such as depression [13] and Alzheimer’s disease [14]. In addition, first studies on the association between IL-6 and brain volume suggested
a role of increased serum levels of IL-6 in brain atrophy during normal aging in conjunction
with other cytokines [15]. In addition, an association between IL-6 levels and decreased hippocampal gray matter
volume in middle-aged adults has recently been reported [16].

However, the hypothesis that IL-6 is mainly proinflammatory and neurodegenerative
is challenged [17] with results supporting that this cytokine has several anti-inflammatory and immunosuppressive
activities that may play a downregulating role in inflammatory conditions [17]. In addition, IL-6 may act as a developmental neurotrophic factor [18,19], and it has been shown to improve survival in vitro of several classes of neurons [20-22]. Moreover, it is suggested that IL-6 predominantly plays a protective role by improving
survival of neurons in culture [21,23,24], protecting neurons from excitotoxic and ischemic insults [25-28], and promoting the growth of axons and consequently the number of synapses in a region
[29-32]. Additionally, evidence shows that IL-6 may play a major role in promoting synaptic
plasticity, LTP, and memory consolidation [33-35]. Furthermore, IL-6 is found to regulate survival of differentiated neurons and the
development of astrocytes [36,37]. Overall, these findings from previous studies suggest that higher IL-6 levels may
play a dual role with both neurodegenerative and neuroprotective biological functions.

The current evidence in humans relies on measures of IL-6 in serum and CSF, whereas
limited research on the influence of genetic variants of IL-6 on brain pathology has
been published. The IL-6 gene is located on chromosome 7p21, and the GG genotype of the frequently studied
IL-6 promoter −174 C/G variation relates to higher levels of IL-6 compared to the CC genotype
[38]. Although this single nucleotide polymorphism (SNP) has received a lot of attention
in research in aging and longevity, the findings are inconclusively showing an association
between the numbers of G alleles either with increased [39] or decreased [38] longevity depending on the study design, ethnicity, lifestyle, and cultural differences.
Additional single nucleotide polymorphisms (SNPs), such as rs1800796, influence IL-6
expression (G allele carriers increase IL-6 plasma levels) [40] and are influenced by the presence of other polymorphisms (e.g., rs2069833, rs2069840)
at this chromosomal locus [41]. However, these other genetic variants of IL-6 have hardly been studied in brain function yet.

Further clarification of the biological role of genetic variants of IL-6 in the human brain is needed to describe its multifunctional effects. In this study,
we investigate the role of the IL-6 gene in brain function and brain morphology by investigating the association between
several genetic variants of interleukin 6 and brain morphology in healthy adult individuals.
While this analysis is conducted in a whole-brain fashion, we expect genetic effects
particularly in the hippocampus (HC) since this brain region has a critical role in
normal brain function and several neuropsychiatric disorders. The HC region is a highly
important structure for memory consolidation, and it has shown a strong susceptibility
to stress and response to cytokines [42]. Specifically, several studies have shown that depression, post-traumatic stress
disorder (PTSD), and childhood maltreatment are associated with smaller hippocampal
volumes [42-44]. The role of genetic inflammatory biomarkers, such as IL-6, in these relationships
is unclear.

This study aims at an improved understanding of the genetic background of the dual
role of IL-6 in brain morphology and the hippocampal structure in particular. We hypothesize
that IL-6 polymorphisms are related to brain gray matter volumes, specifically in the hippocampus.
The analysis will inform future studies in clinical psychiatric populations on the
possible role and selection of genetic variants of IL-6 for the study of hippocampal function in neuropsychiatric disorders.

Material and methods

Subjects. Healthy subjects (N = 303) aged 18–65 of Central European ancestry participated in the study. Data were
pooled from various studies conducted at the Department of Psychiatry, University
of Münster, Germany, all employing the same MRI sequence on the same scanner. All
included subjects were thoroughly investigated by experienced psychologists and were
free from any lifetime history of psychiatric disorders according to DSM-IV criteria
[45], as diagnosed with the SCID interview [46]. Exclusion criteria were scores ≥ 10 on the Beck Depression Inventory (BDI) [47], any neurological abnormalities, history of seizures, head trauma or unconsciousness,
intake of any psychotropic medication, and the usual MRI contraindications. Six subjects
had to be excluded because of anatomical abnormalities (abnormally enlarged ventricles)
or strong movement artifacts discovered in the structural MRI images checked by visual
inspection and identification as extreme outliers in the check data quality function
of the VBM8 Toolbox. The remaining N = 297 scans (mean age 33.4 ± 11.7; N = 124 men, N = 173 women) were clear of such problems. Verbal intelligence was estimated by the
Mehrfachwahl-Wortschatz-Intelligenztest (multiple-choice vocabulary intelligence test;
MWT-B) [48]. See Table 1 for sample characteristics. The study was approved by the Ethics Committee of the
University of Münster. After complete description of the study to the participants,
written informed consent was obtained.

Selection of polymorphisms and genotyping

The presently analyzed sequence of the IL-6 gene comprising about 4.8 kb. We investigated genetic polymorphisms within this region
as well as neighboring 5’- and 3’- segments containing possible gene regulatory elements
including positions between 22,731,750 and 22,738,790 at chromosome 7p21. The investigated
region contains 43 single nucleotide polymorphisms (SNPs) [50]. Applying a tagging SNP approach, we used various techniques to limit the number
of SNPs assessed to the most relevant as follows. Initially, we constructed the linkage
disequilibrium (LD) pattern of the CEPH population of the HapMap Phase II genotype
data to identify tagging SNPs by an aggressive tagging approach (MAF > 1% and r2 > 0.8) using the Gevalt v2 software package [51]. Subsequently, we reduced SNP numbers by assessing the ability of limited numbers
of the tagging SNPs to predict the total SNP population using the Stampa algorithm
[52]. With this approach, 97.08% of the variation in the gene was captured using four
tagging SNPs (rs1800795; rs1800796; rs2069833; rs2069840). The mean r2 of individual tagging SNPs in conjunction with one or more tagged SNPs was 0.991
(see Table 2 for details). While the SNPs rs1800795 and rs1800796 have been shown to directly
regulate IL-6 expression, the other two SNPs (rs2069833, rs2069840) are non-coding
variants [53]. The G allele of marker rs2069840 has shown to be associated with lower IL-6 plasma
concentrations under a dominant model in a recently published cohort study [54].

Genotyping of four tagging IL-6 SNPs was carried out following published protocols applying the multiplex genotyping
assay iPLEX™ for use with the MassARRAY platform [55], yielding an overall genotyping completion rate of 98.9% [4/297 genotyping failures
for rs1800795 and rs2069833 (99.0%), 5/297 for rs1800796 and rs2069840 (98.7%)]. Genotypes
were determined by investigators blinded for the study.

Homogeneity of gray matter images was checked using the covariance structure of each
image with all other images, as implemented in the check data quality function. As
described above, six extreme outliers showing anatomical abnormalities or movement
artifacts were identified and excluded. The modulated gray matter images were smoothed
with a Gaussian kernel of 8-mm FWHW. Group statistics were calculated with second
level models using SPM8. For each SNP a separate full factorial model was conducted
using genotype as the between-subjects factor. Age, education, and gender were added
to the model as nuisance regressors. There was an upgrade of the scanner gradient
system in 2008 (“Master” Gradient System to “Quasar Dual” Gradient System). Although the MRI sequence remained identical before and after the gradient system
upgrade, we additionally modeled the scanner upgrade as regressors of no interest.

To control for multiple statistical testing within the entire brain, we maintained
a cluster-level false-positive detection rate at p < 0.05 using a voxel-level threshold of p < 0.005 with a cluster extent (k) empirically determined by Monte Carlo simulations
(n = 1,000 iterations). This was performed by means of the AlphaSim procedure, which
accounted for spatial correlations between BOLD signal changes in neighboring voxels
[57], implemented in the REST toolbox (http://restfmri.net/forum/index.phpwebcite). The empirically determined cluster thresholds were k = 340 voxels. The anatomical
labeling for the whole-brain data was performed by means of the widely used AAL Toolbox
[58] and additionally by means of the Anatomy Toolbox [59]. The present sample had sufficient power (1-β = 80%) to detect relatively small effect
sizes in a three-group ANOVA (f = 0.17) and in an allele-dose regression (r = 0.14), as calculated with G*Power [60].

Results

rs1800795 (−174 C/G): The whole-brain analysis yielded a strong main effect of genotype [43 CC vs. 150
CG vs. 100 GG), x = 24, y = −10, z = −15; F(2,286) = 8.54, puncorrected = 0.0002; pAlphaSim-corrected = 0.002; cluster size k = 577, effect size f = 0.23 (Figure 1)]. According to the automated anatomical labeling, this cluster was located in the
right hippocampus head, extending to the parahippocampal gyrus and the dorsal parts
of the right amygdala. The Anatomy toolbox yielded similar localizations (peak effect
was found in the cornu ammonis and subiculum area, extending to the laterobasal amygdala).
There were no other areas in the entire brain surviving our corrected statistical
threshold. Repeating this analysis with smoothing kernels of 6 mm or 10 mm still would
yield significant findings.

We further checked for interactions of the rs1800795 genotype and age as well as gender
by modeling the interaction term in the three-group ANOVA model and the allele-dose
regression. However, none of the interactions reached even a trend level of significance.
Thus, the observed genotype effect on hippocampal gray matter volumes was comparable
in men and women, and found across the entire age range.

rs1800796, rs2069833, rs2069840: No significant effects of these SNPs on hippocampus morphometry could be discerned
in the whole-brain analysis.

Discussion

This imaging genetics study investigated the association between the IL-6 gene and brain morphology in a large cohort of healthy adult participants in a whole-brain
analysis approach. Carriers of the G-allele of the IL-6 genetic variant rs1800795 (−174 C/G) showed a significant association with larger hippocampal volumes on the right side
in healthy subjects. This genotype effect was remarkably specific to the hippocampus,
with no other structure surviving our statistical threshold corrected for the entire
brain. The findings are suggestive of a neuroprotective role of the IL-6 gene [rs1800795 (−174 C/G)] on hippocampal morphology. The IL6 genotype effect was
found lateralized to the right. However, at a more lenient uncorrected statistical
threshold, a similar genotype effect in the same direction could also be detected
in the left hippocampus (p = 0.007, uncorrected, in the allele-dose model). Therefore, we discuss the observed
effects for the hippocampus in general. The other investigated three SNPs showed no
significant association with gray matter volume in our study. Since the SNPs 2069840
has been related to reduced IL-6 plasma levels, the lack of association in our study
can be interpreted as consistent with the assumption that reduced plasma levels do
not exert neuroplastic, neuroproliferative, or neuroprotective effects. In contrast,
the marker rs1800796 showed no association with gray matter volume in our study, although,
in a previous study, the G allele of this SNP has also been associated with higher
IL-6 plasma levels [40]. Since these findings were derived from a clinical cohort of patients with diabetic
nephropathy without data on brain morphometry, a direct comparison with our study
is precluded.

Our study shows the strongest association between the IL-6 genetic variant and HC volume, which has a number of critical functions under healthy
and pathological conditions. It is part of a brain network including the dorsomedial
and dorsolateral prefrontal cortex, the anterior cingulate cortex, and the amygdala
dysregulated in major depression [61]. The HC is central to memory impairment, as seen in non-clinical samples [62] as well as in MDD [63]. Because the HC is a highly stress-sensitive brain region [64] and stress (psychological or psychosocial stress) is related to structural changes
in the HC [65-67], atrophy of the HC has been described in imaging studies as a pathological neurobiological
feature of depression associated with stress [68]. A meta-analysis of hippocampal volumes in patients with MDD confirmed that patients
had hippocampal volumes approximately 4–6% smaller than matched control subjects in
the left and right HC [69,70].

Although the possible role of IL-6 in brain morphology has not been extensively studied
yet, our findings are in contrast with previous reports. These show associations between
increased IL-6 plasma levels and reduced hippocampal volume in a relatively small
study (N = 76) of middle-aged, relatively healthy individuals [16] in a study on first-episode psychosis [71], and in two studies investigating various brain areas and total brain volume, respectively,
during aging [72,73]. Except in one study in relatively healthy individuals, these previous studies investigated
individuals with underlying neuropsychiatric conditions. Variation in findings between
these studies may be due to other methodological differences, such as the location
of gray matter volume changes. While volume changes were located in the left HC in
the study by Marsland et al. [16], and in various brain areas and total brain volume in the above-mentioned studies
on aging [72,73], our results were specific to the HC. Another important difference between studies
is the biological model of IL-6 effects in the brain. While those previous studies
explain their findings using an inflammatory model in which it is proposed that IL-6
plays a proinflammatory role, the explanation of our study builds on the proven anti-inflammatory
and immunosuppressive effects of IL-6 according to the well-established dual role
of IL-6 [74]. A possible mechanistic explanation to support our finding that IL-6 was associated
with increased HC volumes relates to the previously reported neuroproliferative effects
of IL-6. For example, it has been shown that cytokines, including IL-6, despite being
large molecules not freely passing through the blood–brain barrier, can enter the
brain via various pathways (humoral, cellular, neural) [75] to exert their biological effects in the brain even under physiological conditions.
More specifically, it has been shown that IL-6 primarily exerts its biological effects
through a hexameric receptor ligand complex including the gp130 receptor [11] and the IL-6 receptor [76]. Distinct regions of gp130 activate specific signal-transduction pathways, such as
the Janus kinase (JAK) signal transducer and activator of transcription (STAT), mitogen-activated
protein kinase (MAPK)/cAMP responsive element-binding protein (CREB), Ras-MAPK, and
PI-3 kinase (for review [77]). These pathways are related to neural plasticity by their ability to induce processes
of neurogenesis, such as gliogenesis, neuronal differentiation, cAMP response element
binding (cAMP), neural progenitor proliferation, and neuronal survival [77-79], and to enhance synaptic plasticity, LTP, and memory consolidation [33-35]. Through activation of these pathways, IL-6 has the ability to exert neuroprotective
and neuroproliferative effects. In addition, IL-6 has been found to regulate survival
of differentiated neurons and the development of astrocytes [36,37]. Some in-vitro studies show IL-6 release by activated microglia is a key inhibitor
of neurogenesis by approximately 50%; others show IL-6 promoting differentiation of
neural stem cells (NSCs) [10,80-82]. NSCs derived from rodent spinal cord show that IL-6 induces NSC proliferation via
the JAK2/STAT3 and MAPK pathways [83]. Supporting a role of IL-6 in neuroproliferation is an in-vivo study showing that IL-6 knockout mice have reduced proliferating NSCs specifically
in the HC, hence underlining the importance of IL-6 in cell proliferation and cell
survival [84].

Despite mechanistic evidence and studies in humans for both pro- and anti-inflammatory
effects of IL-6 in the brain, the role of IL-6 in the hippocampus remains to be clarified.
Specifically, it is questionable that increased levels of IL-6 have purely degenerative
effects since proliferative effects of IL-6 in the HC were demonstrated in an exercise
study in mice: a wheel-running study in mice over 16 weeks showed that exercise increased
IL-6 levels in the HC, whereas other cytokines such as TNF and IL-1ra decreased during
exercise [85]. These results suggest that an upregulation of IL-6 could have anti-inflammatory
effects and be neuroprotective in the cytokine milieu of the HC, and thereby IL-6
may buffer cognitive decline through exercise-induced changes in the HC milieu.

Translating these findings into a human study, one could argue that peripherally increased
IL-6 levels could be interpreted as an anti-inflammatory activity rather than a proinflammatory
state. Hence, previously observed correlations between increased plasma levels of
IL-6 and decreased HC volumes could alternatively be interpreted as an anti-inflammatory
response of IL-6 to other increased cytokines such as TNF and IL-1beta. Indeed, both
cytokines have previously been shown to be associated with hippocampal volumes (TNF)
[86] and with increased white matter hyperintensities (IL-1beta) [87] in healthy individuals. In such a case, IL-6 would only be a marker of a global inflammatory
process, and reduced brain volume might primarily be induced by proinflammatory cytokines
such as TNF and IL1-beta.

In light of these studies suggesting effects of IL-6 on various mechanisms subserving
neuroproliferation and assuming that the carriers of the G-allele of the IL-6 polymorphism rs1800795 (−174 C/G) in our sample have increased IL-6 levels as previously
reported, it can be suggested that in our imaging study, this particular SNP might
exert neuroprotective effects on the HC via increased IL-6 levels, hence the observed
increased gray matter volume.

Our study has strengths and limitations. We were able to employ the genetic analysis
in a large imaging sample using a cohort of carefully selected and well-characterized
healthy individuals. For future studies, clinical measures such as hypertension or
BMI could be useful covariates when investigating genetic inflammatory biomarkers
such as IL-6; however, the relevance of hypertension might be of greater relevance
in clinical samples than in our healthy cohort. Our discussion is based on the assumption
that larger gray matter values in the hippocampus correspond to better function. Albeit
reduced hippocampal volumes are consistently found in neuropsychiatric disorders,
the relation of volume and function remains to be established more firmly. Although
no protein data were available to validate the well-described upregulation of IL-6
by the SNP rs1800795 (−174 C/G), our study is the first genetic study investigating
the association between the IL-6 gene and brain morphometry, and the HC in particular. Future genetic imaging studies
would benefit from additional protein data. Moreover, a clinical control group with
a psychiatric disorder such as depression or psychosis/schizophrenia might add knowledge
on the dual role of the IL-6 gene in health and disease states. Another important consideration for interpreting
these results is related to the lack of a cutoff of level of IL-6 defining normal,
increased, and decreased peripheral IL-6 levels, limiting the interpretation of physiological
and pathological brain conditions. The LD indices indicate complete LD (D’ = 1) for
the correlation of all four marker combinations (except D’ = 0.993 for rs1800795 x
rs2069833), which indicates that the reported findings are not explained by relevant
SNP correlations.

Conclusion

This imaging genetic study suggests the IL-6 genetic variant rs1800795 (−174 C/G) as a biomarker of hippocampal morphometry. This
genetic variant may exert neuroprotective effects on hippocampal volume in healthy
individuals. Replication in independent and clinical samples is warranted.

Competing of interests

KD has received speaker fees from Pfizer, Lilly, and Bristol-Myers Squibb; she has
been a consultant for Johnson & Johnson and has received funding by Astra Zeneca.
All other authors declare no conflicts of interest.

Authors’ contributions

BTB conceived and proposed the genetic analysis, and wrote the first draft together
with UD. UD performed the imaging and imaging-genetics analysis, and drafted parts
of the manuscript. CK contributed to the design of the study, oversaw recruitment
of participants, and contributed to the draft manuscript. DG contributed to the recruitment
and assessment of participants. TS contributed to the study design and the interpretation
of the results. EB contributed to the genetic analyses of the results. PO, SS, AV,
and CU carried out the recruitment, assessment, and measurements of participants.
WH, JB, and HK oversaw and conducted the imaging MRI component of the study. KD contributed
to data of participants, and oversaw DNA collection and contributed intellectually
to a draft of the manuscript. VA contributed intellectually to the content of the
manuscript. UD oversaw, conceived the imaging study, and contributed to the writing
of the MS. All authors contributed intellectually to the manuscripts and approved
the final versions of the manuscript.

Acknowledgements

The study was supported by grants of Innovative Medizinische Forschung (IMF) of the
Medical Faculty of Münster (IMF DA120309 to UD, IMF DA211012 to UD, IMF DA111107 to
UD, and IMF AR510403 to VA), Interdisziplinäres Zentrum für Klinische Forschung (IZKF)
of the Medical Faculty of Münster (IZKF FG04 to CK), and Rolf-Dierichs-Stiftung (ZUW80037
to UD), Germany.